A key problem in early drug discovery is the occurrence of false-positive, promiscuous ligands, which can dominate hit lists. The long-term goal of this research is to understand this promiscuity, with a focus on what may be the dominant mechanism underlying it: compound aggregation followed by receptor sequestration. Recent studies suggest that aggregation may be a property of many molecules including hits, leads, and biological reagents. The goal is to investigate what sorts of molecules act as promiscuous aggregators, to determine how they do so, and to develop simple screens to detect them. The specific aims are: 1. To explore the range of molecules that act as promiscuous aggregators. Promiscuous aggregators can occur among screening hits, leads, biological reagents, and even drugs. How likely are "drug-like molecules" to aggregate and inhibit promiscuously at micromolar and sub-micromolar concentrations? What range of receptors do they inhibit? We will sample screening libraries, well-studied biological reagents, and finally bona fide drugs, for promiscuous aggregators. Aggregating inhibitors have characteristic signatures, and may be identified using: inhibition of unrelated model enzymes, sensitivity to enzyme concentration, incubation, and detergent, and dynamic light scattering. We will also consider rapid, robust assays to detect these inhibitors. Such assays may find wide use as counter-screens in lead discovery. 2. To understand the mechanism of aggregation-based inhibition. Many promiscuous inhibitors aggregate, and most molecules that form large aggregates inhibit promiscuously. Exactly how they do so remains unclear. Do aggregating inhibitors directly associate with enzymes? If so, do they adsorb enzyme or absorb enzyme? Is aggregation-based inhibition reversible? How exactly do the aggregating species sequester enzyme? Can aggregating small molecules actually play a beneficial role in preventing the nucleation and pathological aggregation of proteins? A combination of simple techniques, such as centrifugation, protein gels, and enzymology, and higher resolution techniques, such as electron and fluorescence microscopy, will be used to investigate these questions.